The present invention relates to a novel convergent synthesis of bivalirudin, which is a 20-mer peptide of formulaH-D-Phe1-Pro-Arg-Pro-Gly5-Gly-Gly-Gly-Asn-Gly10-Asp-Phe-Glu-Glu-Ile15-Pro-Glu-Glu-Tyr-Leu20-OH (SEQ ID NO 1)  (I)
The invention further relates to several protected peptides as intermediates in the synthesis of bivalirudin.
Proteolytic processing by thrombin is pivotal in the control of blood clotting. Hirudin, a potential clinical thrombin peptide inhibitor, consists of 65 amino acids. But also shorter peptide segments have proven effective for the treatment of thrombosis, a life threatening condition.
U.S. Pat. No. 5,196,404 discloses, among others, bivalirudin, one of these shorter peptides, which are potent thrombin inhibitors. Bivalirudin is also known as hirulog-8, BG-8967, Efludan, Angiomax® or Hirulog® and possess the amino acid sequence given in formula I.
WO 98/50563 describes a method for the preparation of various peptides, including bivalirudin, by recombinant technology. The method comprises expressing the peptide as part of a fusion protein, followed by release of the peptide from the fusion protein by an acyl acceptor.
Okayama et al. Chem. Pharm. Bull. 1996, 44, 1344-1350, and Steinmetzer et al. Eur. J. Biochem. 1999, 265, 598-605, devise a solid phase synthesis of different hirulogs on Wang resin. The Wang resin requires cleavage of the peptide from the resin with concentrated trifluoroacetic acid. In a similar solid phase synthesis approach for the preparation of bivalirudin, WO 91/02750 discloses a sequential approach of attaching single Boc-protected amino acids to Boc-L-leucine-o-divinylbenzene resin, followed by simultaneous deprotection and detachment using HF/p-cresol/ethyl methyl sulfate and subsequent lyophilisation and purification. In both cases, the cleavage of the peptide from the resin requires aggressive acidic conditions, which are likely to cause concomitant global deprotection and might result in undesired side reactions with amino acid residues, thus negatively affecting product purity. Moreover, side-reactions often arise in solid phase synthesis by misincorporation, double-hits of single amino acids and/or racemization and lead to side-products which have a structure very similar to that of the target peptide. Purification is therefore awkward and results in loss of yield. Especially longer peptides are prone to adopt an irregular conformation while still attached to the solid support, which makes it even more difficult to attach additional amino acids to the growing chain. Therefore, this problem increases as the length of the peptide increases.
WO 2007/033383 discloses a method for the production of bivalirudin based on a solid phase synthesis or a combination of solid phase and solution synthesis (mixed approach). In one embodiment, the bivalirudin peptide sequence is prepared on a hyper acid-labile resin. In another embodiment, bivalirudin is prepared by coupling a side chain protected N-terminal peptide fragment with a side chain protected C-terminal peptide fragment and subsequent deprotection using strongly acidic conditions. In this case, said N-terminal fragments and the precursor of said C-terminal fragment (i.e. peptide sequence minus Leu) are both prepared by solid phase synthesis. One of the disadvantages of this strategy is substantial formation of D-Tyr19-bivalirudin. This impurity is difficult to remove, thus requiring extra efforts, costs and loss in yield to get the purified product. In addition, the amount of purified bivalirudin obtained in the examples of WO 2007/033383 is only in the range of grams, indicating that this approach is not suitable for production of bivalirudin on large scale with good purity.